My laboratory studies the mechanisms that direct organ development and regeneration. Currently, our primary focus is on the development and regeneration of the kidney. The kidney performs several essential physiological jobs. The kidney collects and excretes metabolic waste and also regulates water balance. In addition, the kidney produces hormones that regulate blood production and blood pressure. Because of these and other important functions, the loss of kidney activity has life-threatening consequences. Our long-term goals are to discover new biomedical interventions that can be used to treat or prevent kidney disease.

Kidney diseases represent a growing global healthcare burden: they affect epidemic numbers of children and adults worldwide, and are steadily climbing in incidence. Kidney diseases can be caused by congenital defects, acute and chronic injuries, malignancy, and as a secondary consequence of conditions like diabetes. Progressive damage to the kidney abolishes its functions and causes end-stage-renal disease (ESRD), which requires renal replacement therapy. Current renal replacement strategies can prolong life but have significant limitations: dialysis is grueling for patients, and there is a long wait (often >10 years) for an organ transplant. Taken together, there is a pressing need to identify innovative therapies to treat kidney disease and to identify renoprotective agents that could ameliorate and/or prevent kidney damage in various settings.

While kidney diseases are diverse in origin, many share a common trait: damage to the basic unit of the kidney called the nephron. Human kidneys vary in the number of nephrons they contain, ranging from several hundred thousand to over one million nephrons in a single kidney (Figure 1). Each nephron is an epithelial tube that is highly specialized. At one end of the nephron is a blood filter (or glomerulus) that interacts with the vasculature to collect fluid from the circulation. The collected filtrate then passes through a long tubule that consists of a series of different epithelial cell types, or so-called segments (indicated by colored intervals in Figure 1). Each tubule segment performs discrete tasks in modifying the filtrate by reabsorbing and secreting solutes—jobs that enable the retention of desirable nutrients and export of metabolic wastes. The last portion of the nephron connects to a collecting duct. Nephrons in mammalian kidneys have intricate loops and convolutions and are organized in arbor-like arrays, with the collected waste ultimately channeled into the bladder.

To date, the pathways that control how renal stem cells give rise to nephrons during kidney development are poorly understood. Part of the reason for this is that the architecture and internal location of the kidney pose challenges for studying nephron development and dysfunction in mammalian models like the mouse. Knowledge about how nephron cell types form during nephrogenesis would be a powerful tool in understanding congenital kidney defects and could be useful in the design of regenerative therapies for kidney diseases where nephrons (or particular cells within them) are destroyed.

For some time, there has been experimental evidence that the kidney exhibits a limited capacity to regenerate. Damage to epithelial cells in nephrons can be followed by a local regenerative response in which new nephron epithelial cells are made. However, the molecular pathways that enable this type of nephron regeneration (and others) remain a mystery and their discovery is hampered by the aforementioned limitations of existing mammalian models.

To study how nephrons are made during development and regenerate after injury, we use the zebrafish, Danio rerio. The zebrafish is an outstanding model for kidney research for numerous reasons. First, zebrafish are a vertebrate species and share many similarities with more complex vertebrates like mammals. For example, there is a high degree of conservation between gene function and basic cellular processes between zebrafish and mammals. Zebrafish embryos develop outside the mother and are optically transparent (Figure 2A), enabling the direct visualization of organ development. The embryo forms an anatomically simple kidney that is made up of two nephrons (Figure 2B). Zebrafish nephrons are comprised of segments with epithelial cells that share gene expression signatures and ultrastructural traits with segments in mammalian nephrons (indicated by the shared color in analogous segments between Figures 1 and 2B). Importantly, there is a diverse arsenal of molecular tools now available to the zebrafish researcher, and these enable high-resolution study of cell biology and genetic analysis.

Our research questions fall into two major categories:

(1) KIDNEY DEVELOPMENT RESEARCH:How do renal stem cells arise during development? How are nephrons constructed from renal stem/progenitor precursors? We are seeking to identify the genetic requirements for making renal cells. We perform genetic screens to isolate zebrafish with defects in nephron formation. One benefit of this approach is that we can discover essential genes and signaling pathways that have never been implicated in renal progenitor biology. In addition, we perform expression studies to identify factors expressed by kidney cells during nephrogenesis, then assign their functional roles using reverse genetics to knockdown the gene or overexpression techniques to examine gain-of-function.

(2) KIDNEY REGENERATION RESEARCH: How can damaged nephron components be replaced? Are there renal stem cells that can facilitate treatment of renal diseases? Can differentiated nephron cells be induced to regenerate damaged nephrons? Can the activation of kidney developmental pathways facilitate regeneration? We are using models of nephron injury in the embryo to discover the cell and molecular events that are required for nephron regeneration. We have designed a novel technique using laser ablation in which particular nephron regions are targeted for cell destruction, and are examining how such regions regenerate. Along with our nephron development studies, it is our hope that these lines of inquiry will shed novel insights into the activities of kidney cells and guide the creation of new therapeutics.

Acknowledgements

Research in the Wingert lab is supported by start-up funds provided by the University of Notre Dame College of Science and Department of Biological Sciences, and a gift from the Gallagher Family to support adult stem cell research at Notre Dame. Wingert lab research is supported from external grants from (1) the March of Dimes, Basil O’Connor Starter Scholar Award #5-FY12-75, (2) the National Institutes of Health, from the National Institute of Diabetes and Digestive and Kidney Diseases through Grants K01DK083512 and R01 R01DK100237, and through the NIH Director’s New Innovator Award DP2OD008470.

FIGURE LEGENDS

Figure 1: Composition of the mammalian kidney. (Left) The mammalian kidney is a bean-shaped organ comprised of functional units known as nephrons (enlarged in center). (Right) Nephrons have a segmental anatomy, being comprised of regions of specialized epithelial cells that perform discrete excretory

Figure 2: The zebrafish is a simple, conserved model in which to study kidney biology. (A) Lateral view of a zebrafish embryo two days after fertilization. The embryo is transparent, with limited pigmentation. (B) Lateral view schematic of the embryo, with the location of the kidney shown in purple. (Enlargement) Dorsal view of the embryonic kidney shows a pair of nephrons (top), with segmental anatomy indicated (bottom)